Apoptosis is a regulated program of cell death involving complex interactions between enhancing (pro-apoptotic) and inhibiting (anti-apoptotic) factors. Lethal proteolytic enzymes, called caspases, are activated to cleave vital cell proteins in response to death-inducing signals. Caspase activity can be inhibited by a class of proteins called inhibitors of apoptosis (lAPs). In turn, lAPs can be repressed by a group of proteins containing an lAP-binding motif (IBM proteins). Drosophila Reaper is an IBM containing, potent pro-apoptotic protein that has been shown to disrupt lAP-mediated inhibition of caspases and promote the auto-ubiquitination and proteasomal degradation of lAPs. Recent reports have identified a new domain in Reaper (the GH3 domain) that is essential for both localization of Reaper to the mitochondria and stimulation of lAP auto-ubiquitination/ destabilization. The goal of this proposal is to determine how Reaper mitochondrial targeting is achieved and how this targeting facilitates Reaper-mediated lAP destruction. These questions will be addressed using mammalian and lepidopteran cell cultures, as well as a powerful Xenopus egg extract system for the reconstitution of apoptotic processes. The mitochondria are favorable targets for cell death-inducing therapies in the treatment of apoptosis-deficient diseases such as cancer. Understanding how the GH3 domain of Reaper associates with the mitochondria to affect the apoptotic program, and what factors regulate this response should provide further insight into the role of mitochondria in apoptosis, potentially providing novel avenues for chemotherapy.